-- Anita Schwegmann wrote:
>> Hi there
>> I have been screening a Yeast Two Hybrid cDNA library constructed from
> embryonic mouse forebrains. As yet I have not pulled out any positive
> clones. I have screened the library using different constructs of my
> "bait" plasmid and although my positive controls worked well, I still
> have not had one positive reaction. I find this strange as the Yeast Two
> Hyrbrid system is renowned for its "false positives".Is there anyone out
> there who has made up a Two Hyrid cDNA library from rat or mouse
> forebrains and have had trouble finding positive clones? Any hgelp would
> be much appreciated.
>> Many Thanks,
> Anita
Hi Anita,
though I don't know how complex your library is and how many
transformants you
screened, it seems as your bait protein just doesn't travel into the
nucleus.
For some strange reason this seems to happen. But are you sure that the
interaction partner is somewhere in the library? Or could the
interaction dependent
on a third component or on posttranslational modification?
We have the same case with one of our baits here. We fused the bait with
GFP and
monitored the intracellular localization under the flourescence
microscope.
That showed us perfect nuclear localization, however, interacting
partners where
not yet detected even after screening more than 30 million
transformants.
Interaction was shown before using a biochemical approach. This led us
to the
conclusion that interaction was dependent on a third component.
hope this helps
Ricky
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Frederik Boernke
Research Group of Molecular Plant Physiology
Institute for Plant Genetics and Crop Plant Research (IPK)
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